A small cassette enables conditional gene inactivation by CRISPR/Cas9

Area of Science:

• Molecular Biology
• Gene Editing
• Genetics

Background:

• CRISPR/Cas9 technology facilitates gene knockouts but struggles with conditional inactivation of essential genes.
• Studying essential genes requires precise control over their activity to avoid lethality.

Purpose of the Study:

• To develop a novel method for conditional gene inactivation, particularly for essential genes.
• To create a tool that allows for the precise temporal and spatial control of gene function using CRISPR/Cas9.

Main Methods:

• Development of the DECAI (Degradation based on Cre-regulated Artificial Intron) system, a small 201-nucleotide cassette.
• Insertion of the DECAI cassette into coding exons of target genes via CRISPR/Cas9 and homology-directed repair.
• Activation of gene inactivation using Cre recombinase, leading to splicing disruption and introduction of stop codons.

Main Results:

• The DECAI cassette is efficiently removed by splicing in the 'off-state', leaving no trace.
• Cre recombinase activation cripples the intron, leading to gene disruption via stop codons in the 'on-state'.
• Successful application in human genes (essential and non-essential) with high recovery frequencies (>5%) and confirmed gene expression loss.

Conclusions:

• DECAI provides a versatile and efficient method for conditional gene inactivation.
• This approach is suitable for studying the loss-of-function phenotypes of essential genes, overcoming previous limitations.